The long-term goal of the proposed research is to understand how cells preserve genome integrity during DNA replication. Specifically, this application focuses on the ATR (ATM and rad3-related) signaling pathway. ATR functions at the apex of a DNA damage and replication stress response pathway that is needed every cell division cycle to promote the complete and accurate replication of the genome. Many cancer cells are highly dependent on ATR function for proliferation and viability because of elevated levels of oncogene-induced replication stress and mutations in other genome maintenance pathways. Thus, ATR may be a useful drug target based on a synthetic lethal approach. In this proposal we test a specific model of how ATR promotes replication fork stabilization and repair, define the consequences of acute ATR inhibition on DNA replication and cell fate outcomes, define and characterize new ATR-regulated proteins that act at damaged replication forks, characterize cancer settings in which ATR inhibition might be useful using synthetic lethality, and test a new model of how ATR is regulated by autophosphorylation. This is a focused proposal aimed at understanding the most important and least understood aspects of ATR function. Specific hypotheses and innovative concepts based on preliminary data are tested using advanced biochemical and genetic approaches. In addition, the aims provide opportunities for unexpected discoveries about the mechanisms that maintain the genome during DNA replication and when ATR pathway inhibitors may be useful in the cancer clinic.